1. Field of the Invention
This invention relates to and has among its objects novel compositions for therapeutic use and methods of making them. It is a particular object of this invention to provide pasteurized compositions containing therapeutically active proteins. Further objects of the invention will be evident from the following description wherein parts and percentages are by weight unless otherwise specified.
2. Description of the Prior Art
Many useful blood fractions and blood proteins are obtained from human blood plasma by fractionation according to known techniques such as, for example, the alcohol fractionation method of Cohn described in U.S. Pat. No. 2,390,074 (1945) and the Journal of the American Chemical Society, Vol. 68, page 459 (1946) and the Rivanol.RTM. ammonium sulfate method. The aforementioned methods as well as other variations and techniques are summarized in "The Plasma Proteins", second edition, Volume III, pages 548-550, Academic Press, New York, N.Y. (1977). These blood fractions contain biologically active proteins that possess certain therapeutic qualities. For instance, Factor VIII or antihemophilic factor is useful against hemophilia; plasminogen is a precursor of plasmin for treatment of acute thromboembolic disorders; immune serum globulin (IgG) is employed in the treatment of congenital gamma globulin deficiency, measles, poliomyelitis and hepatitis A and B; fibronectin has been identified as active in treatment of burns, shock, cancer, etc.; antithrombin III is a coagulation inhibitor, cryoprecipitate itself may be used directly for classic hemophilia; Plasma Protein Fraction (human) and albumin are useful in treatment of shock due to burns, crushing injuries, abdominal emergencies, and any other cause producing a predominant loss of plasma fluids and not red cells; immune globulin, intravenous (modified immune serum globulin) is a substitute for immune serum globulin administerable in larger quantities; Factor VIII inhibitor bypassing active (FEIBA) substance described in U.S. Pat. No. 4,160,025 as a blood-coagulation-promoting preparation for Factor VIII inhibitor patients; alpha-1-antitrypsin can be employed in the treatment of emphysema; plasma growth hormone corrects pituitary growth deficiency, somatomedin is useful in correcting growth deficiencies, other immune serum globulins, e.g., IgA, IgD, IgE, and IgM, may be employed to treat various immune protein deficiencies; prealbumin (U.S. Pat. No. 4,046,877) is employed to increase immunologic competence; plasminogen-streptokinase complex (U.S. Pat. No. 4,178,368) can be administered to patients for treatment of thromboembolisms; ceruloplasmin, transferrin, haptoglobin, and prekallikrein have reagent and other uses.
One problem confronting users of plasma, plasma fractions, and compositions containing individual blood proteins is the thermal instability of the therapeutically active proteins contained therein. In many cases, substantial, and sometimes complete, losses of activity are observed if these proteins are heated above physiological temperatures, i.e., above about 40.degree.-45.degree. C. Consequently, these items require special care during preparation and storage to minimize such deactivation.
The thermal instability of the aforementioned proteins renders them unpasteurizable. Therapeutically active proteins isolated from plasma may contain viruses, e.g., hepatitis virus, present in the source material for the protein fraction, namely, blood from a donor. A risk of contracting hepatitis exists, therefore, for those receiving unpasteurized fractions from blood plasma fractionation because the presence of the virus cannot be detected with certainty by any known procedure. In a large number of situations, this risk is outweighed by the detriment to a patient in not receiving the therapeutic plasma fraction as determined by the physician.
Some therapeutically active proteins derived from plasma have been pasteurized successfully. For example, it is well known that albumin can be pasteurized by heating at 60.degree. C. or 64.degree. C. for 10 hours (Gellis et al, J. Clin. Invest., Vol. 27, pages 239-244 [1948]) in the presence of certain stabilizers such as acetyl-tryptophan and sodium caprylate. Individuals receiving this pasteurized material did not contract hepatitis, thus indicating the inactivation of hepatitis virus while retaining the activity of albumin under the afore-described heating conditions. Plasma Protein Fraction (human) is also stabilized during pasteurization by the above method.
A process for pasteurizing plasminogen is disclosed by Baumgarten et al in U.S. Pat. No. 3,227,626. An aqueous preparation containing 0.25-20 milligrams per milliliter (mg/ml) of plasminogen and further containing 0.1-0.5 molar lysine with a pH of 5.3-7.5 was heated at 60.degree. C. for 10 hours. As the patentee states, hepatitis virus was destroyed and the danger of transmitting hepatitis was removed with retention of plasminogen activity. Attempts to pasteurize plasminogen under the above conditions in the absence of lysine resulted in complete destruction of plasminogen cannot be stabilized with N-acetyl-tryptophan and sodium caprylate during pasteurization, nor can albumin and Plasma Protein Fraction (human) be pasteurized in the presence of lysine.
Singher has described a process for treating plasminogen to produce a material that is not contaminated with hepatitis virus (U.S. Pat. No. 2,897,123). In the patented pasteurization technique aqueous solutions of plasminogen are heated at about 60.degree. C. for about 10 hours. The activity of plasminogen is retained if the solutions have a pH in the range not less than 3 nor greater than 6.5 and an ionic strength not greater than 0.3.
Another method for removing hepatitis virus from a biological material is described in U.S. Pat. No. 4,168,300. The material to be treated is contacted with a preparation, which may be agarose gel or beaded polyacrylamide plastic coupled with a variety of hydrophobic ligands. Plasma and albumin were subjected to the above purification technique to remove hepatitis virus.
Aqueous solutions of the enzyme thrombin have been stabilized (Seegers, Arch. Biochem., 1944, Vol. 3, pages 363-367) during heating. at 50.degree. C. in the presence of saturation amounts of certain glycosides. The stabilized solutions were heated at the above temperature for a period of 48 hours or more with minimal loss of activity. On the other hand, Seegers also discloses that glycosides and polyols have only minimal effectiveness in stabilizing the enzyme prothrombin. The reversible denaturation of lysozyme and ribonuclease was studies by Gerlsma et al, Int. J. Peptide Protein Res., Vol. 4, pages 377-383 (1972). The authors found that certain polyhydric alcohols increased somewhat the temperatures at which these enzymes were denatured. Finally, Simpson et al, in J. Am. Chem. Soc., Vol. 75, No. 21, pages 5139-5152 (1953) and Donovan in J. Sci. Fd. Agric., Vol. 28, pages 571-578 (1977) noted that the denaturation temperature of ovalbumin (an egg white protein) was raised slightly in the presence of sucrose in aqueous solutions of the protein. However, Donovan points out that the temperatures of denaturation of ovalbumin and S-ovalbumin are 84.5.degree. C. and 92.5.degree. C., respectively. Furthermore, ovalbumin and S-ovalbumin, as well as the aforementioned enzymes, have no therapeutic activity in treating disorders in humans, whereas blood plasma proteins are therapeutically active. In fact, as mentioned below, proteolytic enzymes deactivate blood plasma proteins.
Singher, in the aforementioned U.S. Patent, lists some methods of destroying hepatitis virus. The least effective of these methods involves the use of either nitrogen mustard or beta-propiolactone. High energy irradiation in appropriate dosage is effective but destroys biological activity when applied to human blood products. Heat is recognized also as effective against hepatitis virus, the preferred treatment being heating the material at 60.degree. C. for 10 hours. Higher temperatures above 70.degree. C. for shorter intervals or lower temperatures for longer intervals have also been tried with successful results. However, it is important to note that higher temperatures are undesirable because of the potential for denaturation of the proteins. Furthermore, lower temperatures for long intervals are to be avoided because various proteolytic enzymes are activated under these conditions, and these activated enzymes cause protein degradation. Also, the use of temperatures lower than 60.degree. C. for pasteurization has not been shown to consistently yield a material that does not contain the infective virus.
As mentioned above, the recognition that heating at 60.degree. C. and 64.degree. C. for 10 hours successfully destroys the hepatitis virus in albumin was made by Gellis et al, supra. Gellis et al proved experimentally that albumin heated under the above conditions did not transmit hepatitis even if hepatitis virus was present prior to pasteurization. However, the author noted that hepatitis virus survived heating at 56.degree. C. for one hour, a temperature usually employed for the inactivation of viruses. Thus, although heating at temperatures of about 56.degree. C. for one hour will deactivate most viruses, hepatitis virus is not inactivated; and materials containing hepatitis virus, which are heated at 56.degree. C. for one hour, cause infection of hepatitis in individuals receiving such materials.